Lymphatic endothelial sphingosine 1-phosphate receptor 1 enhances macrophage clearance via lymphatic system following myocardial infarction

Figures

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  FIGURE 1. The expression of S1pr1 was down-regulated in cardiac lymphatic endothelial cells. (A) Relative mRNA expression levels of S1pr1 in cardiac LECs of mice at 3 days after sham or LAD operation (n = 6). (B) Schematic diagram of generation of mice with LEC-specific loss of S1pr1 allele. (C) Relative mRNA expression levels of S1pr1 in cardiac LECs of WT and Lyve1-Cre-S1pr1flox/wt mice (n = 3). (D) Western-blotting analysis of S1pr1 expression in cardiac LECs from the indicated groups, with its quantification (n = 3). Data are mean ± S.E.M.

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  FIGURE 2. The reduced expression of LEC-S1pr1 deteriorates cardiac function after acute myocardial infarction. (A–D) Representative images of echocardiography of post-MI hearts in WT and Lyve1-Cre-S1pr1flox/wt mice (A) with quantification of left ventricle ejection fraction (LVEF%), left ventricle fractional shortening (LVFS%), and LV mass (mg) (B,D) (n = 3). (E) The ratio of heart weight to bodyweight of the indicated groups (HW/BW, mg/g) (n = 3). Data are mean ± S.E.M. n.s., no statistical significance.

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  FIGURE 3. The reduced expression of LEC-S1pr1 aggravates post-MI pathological cardiac remodeling. (A) Representative images of H&E staining of serial sections from hearts in WT and Lyve1-Cre-S1pr1flox/wt mice at 28 days after MI (n = 13). (B–E) Representative images of Masson’s Trichrome staining of serial sections from hearts in WT and Lyve1-Cre-S1pr1flox/wt mice at 28 days after MI (B), with quantification of the percentage of infarct size and cardiac fibrosis in left ventricle myocardium (C–E) (n = 13). (F) Representative images of WGA staining of hearts in WT and Lyve1-Cre-S1pr1flox/wt mice at 28 days after MI, with quantification of the cross-section cardiomyocyte area in hearts (n = 8). (G) Representative images of isolectin-B4 staining of hearts in WT and Lyve1-Cre-S1pr1flox/wt mice at 28 days after MI, with quantification of capillary density in post-MI myocardium (n = 8). (H) Representative images of Lyve-1 (LEC marker) staining of hearts in WT and Lyve1-Cre-S1pr1flox/wt mice at 28 days after MI, with quantification of capillary lymphatic density in post-MI myocardium (n = 6). Data are mean ± S.E.M. Scale Bars: (A,B), 2 mm; (F–H), 50 μm; n.s., no statistical significance.

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  FIGURE 4. S1pr1 regulates lymphatic endothelial cell migration, proliferation, and angiogenic activity. (A) RT-qPCR analysis of S1PR1 mRNA levels in LECs of the indicated group (n = 3). (B) MTT assay showed the proliferation of cells of the indicated group (n = 3). (C) Scratch wound healing assay showed the cell migration, with quantification of cell migration area (%) of the indicated group (n = 3). (D) Boyden chamber assay showed the cell migration of cells of the indicated group, with quantification of migrated cells of the indicated group (n = 3). (E) Tube formation assay showed S1PR1 shRNA promoted LEC tube formation, with quantification of the number of branch points (n = 3). Control, scramble RNA lentivirus. OE, S1PR1-overexpressing lentivirus. shRNA, S1PR1 shRNA lentivirus. Data are mean ± S.E.M. Scale Bars: (C–E), 100 μm.

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  FIGURE 5. The reduced expression of LEC-S1pr1 retards macrophage clearance in injured hearts. (A) Representative images of immunostaining of Ly6G (neutrophil marker) from hearts in WT and Lyve1-Cre-S1pr1flox/wt at 1 day after MI, with quantification of the number of neutrophils in infarct zone of myocardium of the indicated groups (n = 5). (B) Representative images of immunostaining of F4/80 (macrophage marker) within infarct zone of hearts in WT and Lyve1-Cre-S1pr1flox/wt at 7 days after MI, with quantification of the number of macrophages in myocardium of the indicated groups (n = 6). (C) Representative images of immunostaining of F4/80 (macrophage marker) from hearts in WT and Lyve1-Cre-S1pr1flox/wt at 3 days after MI, with quantification of the number of macrophages in infarct zone of myocardium of the indicated groups (n = 5). (D) Schematic diagram of the adoptive cell transfer approach. EGFP transgenic mice were used as splenic EGFP+ monocyte donors, and recipient WT or Lyve1-Cre-S1pr1flox/wt adult mice received donor EGFP+ monocytes via intramyocardial delivery at the time of LAD ligation and EGFP donor monocyte trafficking was monitored in post-MI hearts and their draining MLNs (n…

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  FIGURE 6. LEC-S1pr1 regulates macrophage trafficking via ERK/CCL2 signaling pathway. (A) The representative images of transwell cell migration assay of THP-1 cell lines, which were co-cultured with condition medium obtained from LECs of the indicated groups, with their quantification of migrated cells (n = 3). (B) RT-qPCR analysis showed the relative mRNA expression levels of different chemokines in the indicated groups (n = 3). (C) RT-qPCR analysis showed the relative Ccl2 and Ccl21 mRNA expression levels in LECs from hearts of the indicated groups at 7 days after MI (n = 3). (D) The representative images of Boyden chamber assay of THP-1 cell lines, which were co-cultured with the conditioned medium obtained from LECs of the indicated groups, with their quantification of migrated THP-1 (n = 3). (E) Western blotting analysis of ERK activation status in LECs treated with S1P in the indicated groups with their quantification of the ratio of p-ERK/total ERK (n = 3). (F) RT-qPCR showed the CCL2 mRNA expression of the indicated groups (n = 3). U0126, ERK inhibitor. Scale bars for (A,C) 100 μm. Data are mean ± S.E.M. n.s., no statistical significance.

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  FIGURE 7. Working model of lymphatic endothelial S1pr1 enhances macrophage clearance via lymphatic system following myocardial infarction.